Anti-cd19 antibodies with reduced immunogenicity

ABSTRACT

Anti-CD19 B4 antibodies with modified variable regions are disclosed. The modified anti-CD19 variable region polypeptides have alterations to one or more framework regions or complementarity determining regions of the heavy chain variable region or light chain variable region, thereby to reduce a T-cell response.

FIELD OF THE INVENTION

The invention relates generally to variable regions of the anti-CD19murine monoclonal antibody B4 light chain and heavy chain modified toreduce their immunogenicity.

BACKGROUND

CD19 is a surface protein found on B cells and on certain cancerouscells derived from B cells, such as many B cell lymphomas. Anti-CD19monoclonal antibodies have been generated in mice, and show some promisein pre-clinical animal models of B cell-derived cancers. However,mouse-derived antibodies are generally immunogenic in humans. A numberof strategies have been developed to alter mouse-derived antibodies tominimize their immunogenicity in humans. One such strategy,chimerization, involves the fusion of mouse variable regions to humanconstant regions. However, the mouse-derived variable region sequencesremaining following chimerization will often be immunogenic. Anothersuch strategy, humanization, involves the replacement of mouse-derivedframework regions (FRs) within the variable regions with the mostclosely related human-derived sequences, with the optional reversion ofcertain amino acids back to the corresponding mouse amino acid in orderto maintain binding activity. However, even humanized antibodies may beimmunogenic, since the antibody complementarity determining regions(CDRs) generally contain B cell epitopes and T cell epitopes that arenon-self. Indeed, even fully human antibodies are immunogenic; this isthe basis for the formation of anti-idiotype antibodies during thecourse of an immune response. All of these problems may apply tomouse-derived anti-CD19 antibodies as they would to any other type ofantibody. Therefore, there is a need for anti-CD19 antibodies withreduced immunogenicity.

SUMMARY OF THE INVENTION

The present invention is directed to an anti-CD19 murine monoclonal B4antibody which has been modified to reduce its immunogenicity incomparison to wild-type B4 antibody. More specifically, the variableregion of the B4 antibody of the invention is modified to removepotential T-cell epitopes. As a result, B4 antibodies of the inventionhave improved biological properties compared to wild-type B4 antibodies.

Accordingly, in one aspect, the invention features an amino acidsequence defining a modified immunoglobulin heavy chain framework regioncomprising amino acid residues 1-30 of SEQ ID NO:22, wherein one or moreof the amino acid residues at positions X5, X12, X19, X20, X23, and X24are as follows: X5 is Q or E, X12 is V or K, X19 is R or K, X20 is L orV, X23 is K, E or D, or X24 is T or A. According to this aspect of theinvention, at least one of the amino acid residues at positions X5, X12,X19, X20, X23, or X24 is not the same amino acid residue as the aminoacid at the corresponding position in the unmodified immunoglobulinheavy chain framework region as set forth in amino acid residues 1-30 ofSEQ ID NO:13. In one embodiment, X23 is E or D.

In another aspect, the invention features an amino acid sequencedefining a modified immunoglobulin heavy chain framework regioncomprising amino acid residues 1-14 of SEQ ID NO:23, wherein one or moreof the amino acid residues at positions X3, X5, X7, and X8, are asfollows: X3 is K or R, X5 is R, T, or A, X7 is G, D, or E, or X8 is Q orK. According to this aspect of the invention, at least one of the aminoacid residues at positions X3, X5, X7, or X8 is not the same as theamino acid at the corresponding position in the unmodifiedimmunoglobulin heavy chain framework region as set forth in amino acidresidues 36-49 of SEQ ID NO:13. In one embodiment, X7 is E or D.

In another aspect, the invention features an amino acid sequencedefining a modified immunoglobulin heavy chain framework regioncomprising amino acid residues 1-39 of SEQ ID NO:24, wherein one or moreof the amino acid residues at positions X6, X10, X26, X29, and X34 areas follows: X6 is K, D, or E, X10 is K, E, or D, X26 is S, D, or E, X29is S or A, or X34 is V or T. According to this aspect of the invention,at least one of the amino acid residues at positions X6, X10, X26, X29,or X34 is not the same as the amino acid at the corresponding positionin the unmodified immunoglobulin heavy chain framework region as setforth in amino acid residues 60-98 of SEQ ID NO:13. In one embodiment,X10 is E or D.

According to another aspect, the invention features an amino acidsequence defining a modified immunoglobulin light chain framework regioncomprising amino acid residues 1-23 of SEQ ID NO:32, wherein one or moreof the amino acid residues at positions X1, X3, X7, X10, X11, and X19are as follows: X1 is Q or D, X3 is V or A, X7 is S or E, X10 is I or T,X11 is M or L, or X19 is V or A. According to this aspect of theinvention, at least one of the amino acid residues at positions X1, X3,X7, X10, X11, or X19 is not the same as the amino acid at thecorresponding position in the unmodified immunoglobulin light chainframework region as set forth in amino acid residues 1-23 of SEQ IDNO:25. In one embodiment, X3 is A and X7 is E. In another embodiment, X1is D, X10 is I, and X11 is L.

In another aspect, the invention features an amino acid sequencedefining a modified immunoglobulin light chain complementaritydetermining region comprising amino acid residues 24-33 of SEQ ID NO:28.

In another aspect, the invention features an amino acid sequencedefining a modified immunoglobulin light chain framework regioncomprising amino acid residues 56-87 of SEQ ID NO:28.

According to another aspect, the invention features an antibody variableregion comprising an amino acid sequence selected from the groupconsisting of SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO:17, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 26, SEQ ID NO: 27, SEQ IDNO: 28, SEQ ID NO: 29, SEQ ID NO: 30, or SEQ ID NO: 31, wherein theantibody variable region specifically binds to CD19.

According to another aspect, the invention features a polypeptide atleast 90% or at least 95% identical to a B4 antibody heavy chainvariable region, the polypeptide comprising an amino acid substitutionat one or more residues corresponding to Val12, Leu20, Lys23, Thr24,Lys38, Gly42, Gln43, Lys65, Lys69, Ser85, Ser88, or Val93. In oneembodiment, the polypeptide comprises one or more of substitutionsGln5Glu, Val12Lys, Arg19Lys, Leu20Val, Lys23Glu, Lys23Asp, Thr24Ala,Lys38Arg, Arg40Thr, Gly42Asp, Gly42Glu, Gln43Lys, Lys65Asp, Lys65Glu,Lys69Glu, Lys69Asp, Ser85Asp, Ser85Glu, Ser88Ala, or Val93Thr.

According to another aspect, the invention features a polypeptide atleast 90% or at least 95% identical to a B4 antibody light chainvariable region, the polypeptide comprising an amino acid substitutionat one or more residues corresponding to Val3, Ser7, Ile10, Met11,Val19, Val29, Ser51, Leu53, Ala54, or Ser75. In one embodiment, thepolypeptide comprises one or more of substitutions Gln1Asp, Val3Ala,Ser7Glu, Ile10Thr, Met11Leu, Val19Ala, Val29Ala, Ser51Asp, Leu53Thr,Ala54Asp, or Ser75Glu.

In another aspect, the invention features a nucleic acid encoding apolypeptide according to any one of the embodiments of the invention.

In another aspect, the invention features a method of treating apatient, the method comprising the step of administering atherapeutically effective amount of a polypeptide according to any oneof the embodiments of the invention to a patient.

In another aspect, the invention features a method for targeting a cellwith CD19 on its surface, the method comprising the step ofadministering an antibody variable region according to any one of theembodiments of the invention. In one embodiment of the method the cellis a tumor cell.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts the nucleic acid sequence encoding B4 antibody heavychain variable region (B4 VH0) (SEQ ID NO:1).

FIG. 2 depicts the nucleic acid sequence encoding an exemplary B4antibody heavy chain variable region incorporating codons for themutations K23E, G42D, K69E, and S85D (B4 VHv1) (SEQ ID NO:2).

FIG. 3 depicts the nucleic acid sequence encoding an exemplary B4antibody heavy chain variable region incorporating codons for themutations K69E, and S85D (B4 VHv2) (SEQ ID NO:3).

FIG. 4 depicts the nucleic acid sequence encoding an exemplary B4antibody heavy chain variable region incorporating codons for themutations Q5E, V 12K, R19K, L20V, T24A, S85D, and S88A (B4 VHv3) (SEQ IDNO:4).

FIG. 5 depicts the nucleic acid sequence encoding an exemplary B4antibody heavy chain variable region incorporating codons for themutations Q5E, R19K, L20V, R40T, Q43K, K65D, S85D, S88A, and V93T (B4VHv4) (SEQ ID NO:5).

FIG. 6 depicts the nucleic acid sequence encoding an exemplary B4antibody heavy chain variable region incorporating codons for themutations Q5E, V 12K, R19K, L20V, T24A, K38R, R40A, Q43K, K65D, S85D,and V93T (B4 VHv5) (SEQ ID NO:6).

FIG. 7 depicts the nucleic acid sequence encoding an exemplary B4antibody heavy chain variable region incorporating codons for themutations Q5E, R19K, L20V, K65D, S85D, and V93T (B4 VHv6) (SEQ ID NO:7).

FIG. 8 depicts the nucleic acid sequence encoding B4 antibody lightchain variable region (B4 VK0) (SEQ ID NO:8).

FIG. 9 depicts the nucleic acid sequence encoding an exemplary B4antibody light chain variable region incorporating codons for themutations V3A, S7E, and A54D (B4 VKv1) (SEQ ID NO:9).

FIG. 10 depicts the nucleic acid sequence encoding an exemplary B4antibody light chain variable region incorporating codons for themutations Q1D, 110T, M11L, and A54D (B4 VKv2) (SEQ ID NO:10).

FIG. 11 depicts the nucleic acid sequence encoding an exemplary B4antibody light chain variable region incorporating codons for themutations I10T, M11L, V19A, V29A, and S75E (B4 VKv3) (SEQ ID NO:11).

FIG. 12 depicts the nucleic acid sequence encoding an exemplary B4antibody light chain variable region incorporating codons for themutations I10T, M11L, V19A, S51D, and L53T (B4 VKv4) (SEQ ID NO:12).

FIG. 13 depicts the amino acid sequence of B4 antibody heavy chainvariable region (B4 VH0) (SEQ ID NO:13).

FIG. 14 depicts the amino acid sequence of an exemplary B4 antibodyheavy chain variable region with the mutations K23E, G42D, K69E, andS85D (B4 VHv1) (SEQ ID NO:14).

FIG. 15 depicts the amino acid sequence of an exemplary B4 antibodyheavy chain variable region with the mutations K69E, and S85D (B4 VHv2)(SEQ ID NO:15).

FIG. 16 depicts the amino acid sequence of an exemplary B4 antibodyheavy chain variable region with the mutations Q5E, V12K, R19K, L20V,T24A, S85D, and S88A (B4 VHv3) (SEQ ID NO:16).

FIG. 17 depicts the amino acid sequence of an exemplary B4 antibodyheavy chain variable region with the mutations Q5E, R19K, L20V, R40T,Q43K, K65D, S85D, S88A, and V93T (B4 VHv4) (SEQ ID NO:17).

FIG. 18 depicts the amino acid sequence of an exemplary B4 antibodyheavy chain variable region with the mutations Q5E, V12K, R19K, L20V,T24A, K38R, R40A, Q43K, K65D, S85D, and V93T (B4 VHv5) (SEQ ID NO:18).

FIG. 19 depicts the amino acid sequence of an exemplary B4 antibodyheavy chain variable region with the mutations Q5E, R19K, L20V, K65D,S85D, and V93T (B4 VHv6) (SEQ ID NO:19).

FIG. 20 depicts the amino acid sequence of an exemplary B4 antibodyheavy chain variable region with the mutations V 12K, K23E, G42D, K65D,K69E, and S85D (B4 VHv11) (SEQ ID NO:20).

FIG. 21 depicts the amino acid sequence of an exemplary B4 antibodyheavy chain variable region with the mutations Q5E, V 12K, R19K, L20V,T24A, R40T, Q43K, K65D, S85D, S88A, and V93T (B4 VHv34) (SEQ ID NO:21).

FIG. 22 depicts the amino acid sequence of an exemplary B4 antibodyheavy chain framework region with undefined amino acid residues X5, X12,X19, X20, X23, and X24 (B4 VHfr1) (SEQ ID NO:22).

FIG. 23 depicts the amino acid sequence of an exemplary B4 antibodyheavy chain framework region 2 with undefined amino acid residues X3,X5, X7, and X8 (B4 VHfr2) (SEQ ID NO: 23)

FIG. 24 depicts the amino acid sequence of an exemplary B4 antibodyheavy chain framework region 3 with undefined amino acid residues X6,X10, X26, X29, and X34 (B4 VHfr3) (SEQ ID NO:24).

FIG. 25 depicts the amino acid sequence of B4 antibody light chainvariable region (B4 VK0) (SEQ ID NO:25).

FIG. 26 depicts the amino acid sequence of an exemplary B4 antibodylight chain variable region with the mutations V3A, S7E, and A54D (B4VKv1) (SEQ ID NO:26).

FIG. 27 depicts the amino acid sequence of an exemplary B4 antibodylight chain variable region with the mutations Q1D, 110T, M11L, and A54D(B4 VKv2) (SEQ ID NO:27).

FIG. 28 depicts the amino acid sequence of an exemplary B4 antibodylight chain variable region with the mutations I10T, M11L, V19A, V29A,and S75E (B4 VKv3) (SEQ ID NO:28).

FIG. 29 depicts the amino acid sequence of an exemplary B4 antibodylight chain variable region with the mutations I10T, M11L, V19A, S51D,and L53T (B4 VKv4) (SEQ ID NO:29).

FIG. 30 depicts the amino acid sequence of an exemplary B4 antibodylight chain variable region with the mutations V3A, S7E, V19A, A54D, andS75E (B4 VKv11) (SEQ ID NO:30).

FIG. 31 depicts the amino acid sequence of an exemplary B4 antibodylight chain variable region with the mutations I10T, M11L, V19A, V29A,S51D, L53T, and S75E (B4 VKv34) (SEQ ID NO:31).

FIG. 32 depicts the amino acid sequence of an exemplary B4 antibodylight chain framework region with undefined amino acid residues X1, X3,X7, X10, X11, and X19 (B4 VKfr1) (SEQ ID NO:32).

FIG. 33 depicts the amino acid sequence of an exemplary B4 antibodylight chain complementarity determining region with undefined amino acidresidues X3, X5, and X6 (B4 VKcdr2) (SEQ ID NO:33).

FIG. 34 depicts the amino acid sequence of B4 antibody heavy chainvariable region. The complementarity determining regions are underlined.The modifiable amino acid residues are shown in bold.

FIG. 35 depicts the amino acid sequence of B4 antibody light chainvariable region. The complementarity determining regions are underlined.The modifiable amino acid residues are shown in bold.

FIG. 36 is an amino acid sequence alignment of B4 antibody heavy chainvariable regions VH0 (SEQ ID NO:13), VHv1 (SEQ ID NO:14), VHv2 (SEQ IDNO:15), VHv3 (SEQ ID NO:16), VHv4 (SEQ ID NO:17), VHv5 (SEQ ID NO:18),VHv11 (SEQ ID NO:20), and VHv34 (SEQ ID NO:21).

FIG. 37 is an amino acid sequence alignment of B4 antibody light chainvariable regions VK0 (SEQ ID NO:25), VKv1 (SEQ ID NO:26), VKv2 (SEQ IDNO:27), VKv3 (SEQ ID NO:28), VKv4 (SEQ ID NO:29), VKv11 (SEQ ID NO:30),and VKv34 (SEQ ID NO:31).

FIG. 38 shows the results of an ADCC assay on Daudi Burkitt's lymphomacells performed with B4 VHv4/VKv4 antibody expressed either from HEK293T cells (empty triangles) or from YB2/0 cells (filled triangles), andB4 VHv5/VKv4 antibody expressed either from a NS/0 cell line (emptycircles) or from YB2/0 cells (filled circles) as described in Example 4.

FIG. 39 shows the results of treatment of mice transplanted with humanPBMCs treated with either the B4 VHv4/VKv4 antibody of the invention(striped bars), Leu 16 antibody (white bars) or PBS (black bars) asdescribed in Example 5.

FIG. 40 shows the results of treatment of mice carrying Namalwa lymphomacells treated with either the B4 VHv4/VKv4 antibody of the invention(empty circles) or PBS (stars) as described in Example 6

FIG. 41 shows the results of treatment of mice carrying Daudi Burkitt'slymphoma cells treated with either the B4 VHv4/VKv4 antibody of theinvention (empty triangles), cyclophosphamide (empty squares), acombination of the B4 VHv4/VKv4 antibody and cyclophosphamide (emptycircles), or PBS (stars) as described in Example 7.

FIGS. 42 a-c show the results of treatment of mice carrying Namalwalymphoma cells with an antibody of the invention combined with variouschemotherapy agents, as described in Example 8. In FIG. 42( a)treatments are cyclophosphamide (empty squares), the B4 VHv4NKv4antibody (X), a combination of the B4 VHv4/VKv4 antibody andcyclophosphamide (filled squares), or PBS (stars). In FIG. 42( b)treatments are vincristine (empty triangles), the B4 VHv4/VKv4 antibody(X), a combination of the B4 VHv4NKv4 antibody and vincristine (filledtriangles), or PBS (stars). In FIG. 42( c) treatments are doxorubicin(empty circles), the B4 VHv4/VKv4 antibody (X), a combination of the B4VHv4/VKv4 antibody and doxorubicin (filled circles), or PBS (stars).

DETAILED DESCRIPTION OF THE INVENTION

The invention is directed to B4 proteins that have reducedimmunogenicity as compared to wild-type B4, as well as methods formaking and using such proteins. More specifically, the inventionprovides mutations within a B4 antibody that have the effect of reducingthe immunogenicity of a B4 antibody itself, primarily by removing T-cellepitopes within B4 that may stimulate to an immune response.

The present invention is directed to a set of modified antibody heavychain (VH) and light chain (VK) variable regions of the anti-CD19 murineantibody B4 (Nadler et al., (1983) J. Immunol. 130:2947-2951; Roguska etal., (1994) Proc. Natl. Acad. Sci. USA 91:969-973), which herein aregenerically termed “B4 VHvx” and “B4 VKvy”, respectively. For reference,the sequence of the heavy chain variable region of the original murineB4 antibody (B4 VH0) and the sequence of the light chain variableregions of the original murine B4 antibody (B4 VK0), with the CDRsunderlined, are provided in FIGS. 34 and 35, respectively.

As compared to the original B4 VH0 and B4 VK0 polypeptides, B4 VHvx andB4 VKvy polypeptides have reduced immunogenicity. More specifically, theinvention provides mutations within B4 VH and/or B4 VK which have theeffect of reducing the immunogenicity of B4 variable regionpolypeptides, primarily by removing T-cell epitopes within thesepolypeptides that may stimulate an immune response. According to theinvention, protein compositions containing the modified forms of the B4variable regions are less immunogenic when administered to a human, butare still competent to specifically bind CD19 and to target cellsexpressing CD19.

As used herein, the terms “Complementarity-Determining Regions” and“CDRs” are understood to mean the hypervariable regions or loops of animmunoglobulin variable region that interact primarily with an antigen.The immunoglobulin heavy chain variable region (VH) and immunoglobulinlight chain variable region (VK) both contain three CDRs interposedbetween framework regions, as shown in FIGS. 34 and 35, respectively.For example, with reference to the amino acid sequence defining theimmunoglobulin heavy chain variable region of the B4 antibody as shownin FIG. 34 (SEQ ID NO:13), the CDRs are defined by the amino acidsequences from Ser31 to His 35 (CDR1), from Glu50 to Asn59 (CDR2), andfrom Gly99 to Tyr109 (CDR3). With reference to the amino acid sequencedefining the immunoglobulin light chain variable region of the B4antibody as shown in FIG. 35 (SEQ ID NO: 25), the CDRs are defined bythe amino acid sequences from Ser24 to His33 (CDR1), from Asp49 to Ser55(CDR2), and from His88 to Thr94 (CDR3).

As used herein, the terms “Framework Regions” and “FRs” are understoodto mean the regions of an immunoglobulin variable region adjacent to theComplementarity-Determining Regions. The immunoglobulin heavy chainvariable region (VH) and immunoglobulin light chain variable region (VK)each contain four FRs, as shown in FIGS. 34 and 35. For example, withreference to the amino acid sequence defining the immunoglobulin heavychain variable of the of the B4 antibody as shown in FIG. 34 (SEQ ID NO:13), the FRs are defined by the amino acid sequences from Gln1 to Thr30(FR1), from Trp36 to Gly49 (FR2), from Tyr60 to Arg98 (FR3), and fromTrp110 to Ser120 (FR4). With reference to the amino acid sequencedefining the immunoglobulin light chain variable region of the B4antibody as shown in FIG. 35 (SEQ ID NO: 25), the FRs are defined by theamino acid sequences from Gln1 to Cys23 (FR1), from Trp34 to Tyr48(FR2), from Gly56 to Cys87 (FR3), and from Phe95 to Lys104 (FR4).Furthermore, amino acid residues depicted in bold in FIGS. 34 and 35 areamino acid residues that may be mutated according to various embodimentsof the invention.

T-cell epitopes can be identified by a variety of computer andnon-computer methods, including predictions based on structure-basedcomputer modeling or by synthesis of peptides and testing for binding tospecific MHC Class II molecules or in an immunogenicity assay. Accordingto the invention, a potential T-cell epitope is a sequence that, whenconsidered as an isolated peptide, is predicted to bind to an MHC ClassII molecule or an equivalent in a non-human species. A potential T-cellepitope is defined without consideration of other aspects of antigenprocessing, such as the efficiency of protein uptake intoantigen-presenting cells, the efficiency of cleavage at sites in anintact protein to yield a peptide that can bind to MHC Class II, and soon. Thus, the set of T-cell epitopes that are actually presented on MHCClass II after administration of a protein to an animal is a subset ofthe potential T-cell epitopes. According to the invention, a T-cellepitope is an epitope on a protein that interacts with an MHC class IImolecule. Without wishing to be bound by theory, it is understood that aT-cell epitope is an amino acid sequence in a protein that failed toundergo the negative T-cell selection process during T-cell developmentand therefore will be expected to be presented by an MHC Class IImolecule and recognized by a T-cell receptor.

According to one embodiment, the invention provides methods related toreducing the immunogenicity of B4 VH and B4 VK regions. According to oneembodiment of the invention, potential non-self T-cell epitopes areidentified in sequences of B4 VH or B4 VK. For example, potentialnon-self T-cell epitopes are identified by computational methods basedon modeling peptide binding to MHC Class II molecules. Substitutions tospecific amino acid residues are then made such that the ability ofpeptides containing potential T-cell epitopes to bind to MHC Class II isreduced or eliminated.

Modified Protein Sequences of Variable Regions of the Invention.

According to one embodiment, the effect of a specific amino acidmutation or mutations is predicted based on structure-based computermodeling. For example, ProPred(http://www.imtech.res.in/raghava/propred; Singh and Raghava (2001)Bioinformatics 17:1236-1237) is a publically available web-based toolthat can be used for the prediction of peptides that bind HLA-DRalleles. ProPred is based on a matrix prediction algorithm described bySturniolo for a set of 50 HLA-DR alleles (Sturniolo et al., (1999)Nature Biotechnol. 17:555-561). Using such an algorithm, various peptidesequences were discovered within B4 VH and B4 VK which are predicted tobind to multiple MHC class II alleles and are therefore likely to beimmunogenic. These peptide sequences and their predicted bindingfrequency to HLA-DR alleles are shown in Table 1.

With reference to Table 1, the sequence of each 9-mer peptide that bindsto at least 5 HLA-DR alleles is indicated, along with its position (#)in the B4 VH region (left column) or B4 VK region (right column). “Bindfreq.” refers to the number of alleles, out of a possible 50 alleles,that the peptide binds, above an arbitrary binding threshold, in thiscase 20%. A binding frequency of “+” indicates the peptide binds to 5-9alleles, “++” indicates the peptide binds to 10-19 alleles, and “+++”indicates the peptide binds to 20-50 alleles. The 20% binding thresholdis relative to a theoretical maximum binding score, as calculated by analgorithm as described by Sturniolo et al.

TABLE 1 Selected peptides of B4 V regions predicted to bind human HLA-DR alleles. VH T cell VK T cell epitopes bind epitopesbind (start pos.) freq. (start pos.) freq.  (2) VQLQQPGAE + (2) IVLTQSPAI +++ (12) VKPGASVRL +  (3) VLTQSPAIM ++ (18) VRLSCKTSG +++(19) VTMTCSASS + (36) WVKQRPGQG + (29) VNYMHWYQQ + (60) YNQKFKGKA +(46) WIYDTSKLA ++ (64) FKGKAKLTV +++ (47) IYDTSKLAS + (80) YMEVSSLTS ++(93) VYYCARGSN +

These potentially immunogenic sequences in the B4 VH and B4 VKpolypeptides can be rendered less immunogenic by introducing specificmutations that reduce or eliminate the binding of a particular peptideto a human HLA-DR allele (see, for example WO98/52976 and WO00/34317).Alternatively, non-human T-cell epitopes are mutated so that theycorrespond to human self epitopes that are present in human germlineantibodies (see for example U.S. Pat. No. 5,712,120).

Guidance for selecting appropriate mutations may be obtained byreference to tertiary and quaternary structure of antibody variableregions. Crystal structures of antibody variable domains are known inthe art and it is found that structures of the FRs are generally verysimilar to one another. A theoretical model of the antibody variableregion of anti-CD19 antibody B4 VH0/VK0 can be constructed from the mostclosely related antibody heavy and light chain variable regions forwhich a structure has been determined, which can be identified by from aprimary structure alignment (Altschul et al., (1990) J. Mol. Biol.215:403-415). A threading algorithm is used to model the B4 light andheavy chains onto the solved structures (Marti-Renom et al., (2000) AnnuRev Biophys Biomol Struct 29:291-325), and the threaded structures maybe further refined to obtain stereochemically favorable energies (Weineret al., (1984) J Am Chem Soc 106:765-784). It was found that the solvedheavy chain and light chain structures designated respectively by theirPDB database accession codes 1FBI (Fab fragment of monoclonal antibodyF9.13.7) and 1MIM (Fab fragment of anti-CD25 chimeric antibody SdzChi621), were useful reference structures for this purpose.

Preferred mutations do not unduly interfere with protein expression,folding, or activity. According to the invention, amino acids atpositions Q5, V12, R19, L20, K23, T24, K38, R40, G42, Q43, K65, K69,S85, S88, or V93 in B4 V11 and amino acids at positions Q1, V3, S7, I10,M11, V19, V29, S51, L53, A54, or S75 in B4 VK can be mutated while stillretaining the ability of the antibody to be expressed and to bind toCD19 at levels comparable to the unmodified form of B4. Thus theinvention encompasses B4 antibodies having at least one modification inthe VH sequence selected from the group of amino acid positionsconsisting of Q5, V12, R19, L20, K23, T24, K38, R40, G42, Q43, K65, K69,S85, S88, and V93 and/or having at least one modification in the VKsequence selected from the group of amino acid positions consisting ofQ1, V3, S7, I10, M11, V19, V29, S51, L53, A54, and S75.

A nonexhaustive list of specific positions found to tolerate amino acidsubstitutions according to the invention is presented in Table 2,together with exemplary substitutions at those positions.

TABLE 2 Substitutions in B4 V regions. Position in B4 VH SubstitutionPosition in B4 VK Substitution Gln5 Glu Gln1 Asp Val12 Lys Val3 AlaArg19 Lys Ser7 Glu Leu20 Val Ile10 Thr Lys23 Glu, Asp Met11 Leu Thr24Ala Val19 Ala Lys38 Arg Val29 Ala Arg40 Ala, Thr Ser51 Asp Gly42 Asp,Glu Leu53 Thr Gln43 Lys Ala54 Asp Lys65 Asp, Glu Ser75 Glu Lys69 Glu,Asp Ser85 Asp, Glu Ser88 Ala Val93 Thr

One set of embodiments includes amino acid substitutions in the B4 VHpolypeptide, selected from Q5E, V12K, R19K, L20V, K23E, T24A, K38R,R40T, G42D, Q43K, K65D, K69E, S85D, S88A, and V93T. Additionallycontemplated substitutions are K23D, G42E, K65E and K69D. Particularcombinations of mutations are also found to be useful. For example, inone specific embodiment, the substitutions K23E and K69E are included.In another specific embodiment, additionally the substitutions G42D andS88A are included, as shown, for example, for B4 VHv1 (SEQ ID NO: 14).In yet another specific embodiment, VHv1 additionally includes thesubstitutions V12K and K65D (B4 VHv11) (SEQ ID NO:20). In a furtherspecific embodiment the substitutions Q5E, V12K, R19K, L20V, S85D, andS88A are included, as exemplified by B4 VHv3 (SEQ ID NO:16). In yet afurther specific embodiment the substitutions Q5E, R19K, L20V, R40T,Q43K, K65D, S85D, S88A, and V93T are included, as exemplified by B4 VHv4(SEQ ID NO:17). In yet a further specific embodiment the substitutionsQ5E, R19K, L20V, K38R, R40A, Q43K, K65D, S85D, and V93T are included, asexemplified by B4 VHv5 (SEQ ID NO:18). In another specific embodiment,substitutions of B4 VHv3 and B4 VHv4 are combined, as shown in thesequence of B4 VHv34 (SEQ ID NO:21).

Another set of embodiments includes amino acid substitutions in the B4VK polypeptide, selected from Q1D, V3A, S7E, 110T, M11L, V19A, V29A,S51D, L53T, A54D, and S75E. In one specific embodiment, the substitutionA54D is included. Particular combinations of mutations are also found tobe useful. For example, in more specific embodiments, additionally thesubstitutions V3A and S7E are included, as exemplified by B4 VKv1 (SEQID NO:26), or the substitutions Q1D, I10T, and M11L are included, asexemplified by B4 VKv2 (SEQ ID NO:27). In a further embodiment, B4 VK1additionally includes the substitutions V19A and S75E, as exemplified inB4 VKv11 (SEQ ID NO:30). In yet another embodiment, the substitutionsI10T, M11L, V19A, V29A, and S75E are included, as exemplified by B4 VKv3(SEQ ID NO:28). In yet a further specific embodiment, the substitutionsI10T, M11L, V19A, S51D, and L53T are included, as exemplified by B4 VKv4(SEQ ID NO:29). In another specific embodiment, substitutions of B4 VKv3and B4 VKv4 are combined, as shown in the sequence of B4 VKv34 (SEQ IDNO:31).

A primary structure alignment of some exemplary sequences of B4 VHvx andB4 VKvx of the invention, described above, are presented in FIGS. 36 and37, respectively. Amino acids depicted in bold are positions of VH0 andVK0 that may be mutated according to the invention, and underlined aminoacids represent CDRs. VHv1-VHv34 and VKv1-VKv34 are representative heavyand light chains, respectively, with specific amino acid substitutionsthat reduce immunogenicity.

Variable region compositions of the invention include at least a heavychain or a light chain of the invention. For example, in one embodiment,the variable region contains B4 VHv1 (SEQ ID NO:14) and B4 VK0 (SEQ IDNO:25). In another embodiment, the variable region contains B4 VHv1 (SEQID NO:14) and B4 VKv1 (SEQ ID NO:26). In yet another embodiment, thevariable region contains B4 VHv4 (SEQ ID NO:17) and B4 VKv4 (SEQ IDNO:29). In yet another embodiment, the variable region contains B4 VHv5(SEQ ID NO:23) and B4 VKv4 (SEQ ID NO:29). It is appreciated that otherembodiments of the invention are easily obtained by combinatoriallymatching the complete set of B4 VHvx and B4 VKvy polypeptidescontemplated by the invention. Useful combinations are furtherdetermined experimentally, by analyzing protein compositions containingthese combinations, such as a B4 VHvx/VKvy antibody, for theirexpressibility and CD-19 binding activity, as well as their reducedimmunogenicity, as described in more detail below.

Verification of the Reduced Immunogenicity of Variable Regions of theInvention.

To verify that a mutation of the invention has indeed resulted inreduced immunogenicity, standard experimental tests, which are wellknown in the art, can be employed. For example, a T-cell stimulationassay may be used (e.g. Jones et al., (2004), J. Interferon CytokineRes., 24:560). In such an assay, human peripheral blood mononuclearcells (PBMCs) are obtained and cultured according to standardconditions. After an optional pre-stimulation, a peptide correspondingto a potential MHC Class II epitope is added to the culture of PBMCs;the PBMCs are further incubated, and at a later time tritiated thymidineis added. The peptide can be a minimal 9-mer, or can have about 10 to15, or more than 15, amino acids. After further incubation of the cells,incorporation of tritiated thymidine into DNA is then measured bystandard techniques.

The T-cell stimulation assay is thought to work by the followingmechanisms. First, if a peptide is used as a stimulator, the peptidemust first bind to an MHC Class II molecule present on a cell among thePBMCs. Second, the MHC Class II/peptide complex must interactproductively with a T-cell receptor on a CD4+ T-cell. If the testpeptide is unable to bind sufficiently tightly to an MHC Class IImolecule, no signal will result. If the peptide is able to bind an MHCClass H molecule and there are T-cells expressing an appropriatelyrearranged T-cell receptor capable of recognizing a particular MHC ClassII/peptide complex, a signal should result. However, if such T-cellshave been deleted as a result of a negative selection process, no signalwill result. These mechanisms are considered relevant to theimmunogenicity of a protein sequence, as inferred from the stimulationor lack of stimulation by a given peptide.

If recognizing T-cells are present in very low numbers in the PBMCpopulation for stochastic reasons relating to failure of an appropriateT-cell receptor to take place or proliferation of other, unrelatedT-cells followed by homeostasis of the T-cell population, there may alsobe no signal even though a signal is expected. Thus, false negativeresults may occur. Based on these considerations, it is important to usea large number of different sources of PBMCs and to test these samplesindependently. It is also generally useful to test PBMCs from anethnically diverse set of humans, and to determine the MHC Class IIalleles present in each PBMC population.

The standard T-cell assay has the disadvantage that the tritiumincorporation signal is often only two-fold greater than the backgroundincorporation. The proteins and peptides of the invention may also betested in a modified T-cell assay in which, for example, purified CD4+T-cells and purified dendritic cells are co-cultured in the presence ofthe test peptide, followed by exposure to tritiated thymidine and thenassayed for tritiated thymidine incorporation. This second assay has theadvantage that tritiated thymidine incorporation into irrelevant cells,such as CD8+ T-cells, is essentially eliminated and background is thusreduced.

A third assay involves the testing of a candidate protein with reducedimmunogenicity in an animal such as a primate. Such an assay wouldgenerally involve the testing of a B4 VHvx/VKvy protein composition,such as an antibody, that had been designed by testing individualcomponent peptides for potential immunogenicity in a cell-based assaysuch as one described above. Once such a candidate B4 VHvx/VKvy proteincomposition is designed and expressed, the protein is tested forimmunogenicity by injection into an animal.

Injection of the B4 VHvx/VKvy protein composition is generally performedin the same manner as the anticipated route of delivery duringtherapeutic use in humans. For example, intradermal, subcutaneous,intramuscular, intraperitoneal injection or intravenous infusion may beused. If more than one administration is used, the administrations maybe by different routes.

For immunogenicity testing purposes, it may be useful to coadminister anadjuvant to increase the signal and minimize the number of animals thatneed to be used. If an adjuvant is used, it is possible to use anadjuvant lacking a protein component, such as DNA with unmethylated CpGdinucleotides, bacterial lipid A, N-formyl methionine, or otherbacterial non-protein components. Without wishing to be bound by theory,the rationale for avoiding protein-containing adjuvants is that otherproteins may provide T-cell epitopes that will ultimately contribute toan antibody response against the candidate protein.

After one or more administrations of the candidate B4 VHvx/VKvy proteincomposition, the presence of anti-idiotype antibodies is testedaccording to standard techniques, such as the ELISA method. It is foundthat the B4 VHvx/VKvy protein compositions of the invention induceantibody formation less frequently, and to a lesser extent, thancorresponding molecules containing original B4 VH/VK sequences.

Other Configurations of the Variable Regions of the Invention.

In addition to the use of the V regions of the invention in a nakedantibody, it is also possible to configure the V regions of theinvention into antibody fusion proteins that target toxins, immunestimulators, and other proteins, as well as in Fabs, single-chain Fvs,bispecific antibodies, and other configurations known in the art ofantibody engineering.

In certain embodiments of the invention, the light chain variable regionand the heavy chain variable region can be coupled, respectively, to alight chain constant region and a heavy chain constant region of animmunoglobulin. The immunoglobulin light chains have constant regionsthat are designated as either kappa or lambda chains. In a particularembodiment of the invention, the light chain constant region is a kappachain. The heavy chain constant regions, and various modification andcombinations thereof are discussed below in more detail.

Fc Portion

The antibody variable domains of the present invention are optionallyfused to an Fc portion. As used herein, the Fc portion encompassesdomains derived from the heavy chain constant region of animmunoglobulin, preferably a human immunoglobulin, including a fragment,analog, variant, mutant or derivative of the constant region. Theconstant region of an immunoglobulin heavy chain is defined as anaturally-occurring or synthetically produced polypeptide homologous toat least a portion of the C-terminal region of the heavy chain,including the CH1, hinge, CH2, CH3, and, for some heavy chain classes,CH4 domains. The “hinge” region joins the CH1 domain to the CH2-CH3region of an Fc portion. The constant region of the heavy chains of allmammalian immunoglobulins exhibit extensive amino acid sequencesimilarity. DNA sequences for these immunoglobulin regions are wellknown in the art. (See, e.g., Gillies et al. (1989) J. Immunol. Meth.125:191).

In the present invention, the Fc portion typically includes at least aCH2 domain. For example, the Fc portion can include the entireimmunoglobulin heavy chain constant region (CH1-hinge-CH2-CH3).Alternatively, the Fc portion can include all or a portion of the hingeregion, the CH2 domain and the CH3 domain.

The constant region of an immunoglobulin is responsible for manyimportant antibody effector functions, including those mediated by Fcreceptor (FcR) binding and by complement binding. There are five majorclasses of the heavy chain constant region, classified as IgA, IgG, IgD,IgE, and IgM, each with characteristic effector functions designated byisotype.

IgG, for example, is separated into four γ isotypes: γ1, γ2, γ3, and γ4,also known as IgG1, IgG2, IgG3, and IgG4, respectively. IgG moleculescan interact with multiple classes of cellular receptors including threeclasses of Fcγ receptors (FcγR) specific for the IgG class of antibody,namely FcγRI, FcγRII, and FcγRIII. The sequences important for thebinding of IgG to the FcγR receptors have been reported to be in the CH2and CH3 domains.

Widely recognized effector functions of antibodies, particularly of theIgG class, include complement-dependent cytotoxicity (CDC) andantibody-dependent cellular cytotoxicity (ADCC). All of the IgGsubclasses (IgG1, IgG2, IgG3, IgG4) mediate CDC and ADCC to some extent,with IgG1 and IgG3 being most potent for both activities (Chapter 3,Table 3 in Paul, Essential Immunology 4.sup.th Ed., p. 62). CDC isbelieved to occur by multiple mechanisms; one mechanism is initiatedwhen an antibody binds to an antigen on a cell's surface. Once theantigen-antibody complex is formed, the C1q molecule is believed to bindthe antigen-antibody complex. C1q then cleaves itself to initiate acascade of enzymatic activation and cleavage of other complementproteins which then bind the target cell surface and facilitate itsdeath through, for example, cell lysis and/or ingestion by a macrophage.ADCC is believed to occur when Fc receptors on cytotoxic cells, such asnatural killer (NK) cells, macrophages and neutrophils, bind to the Fcregion of antibodies bound to antigen on a cell's surface. Fc receptorbinding signals the cytotoxic cell to kill the target cell.Characteristically, NK cells, believed to be the primary mediators ofADCC, express only FcγRIIIa.

It is often useful to alter the effector functions of an antibody. Forexample, to treat cancers associated with a B cell malignancy or totreat autoimmune diseases with a B cell component, it is useful toenhance the ADCC activity of the antibody. It may be particularly usefulto enhance the ADCC activity of an antibody directed to B cell surfaceantigens present at relatively low density (Niwa et al., (2005) Clin.Cancer Res. 11:2327-2336), such as to CD19. It is believed that theantigen density of CD19 relative to CD20 on the surface of B cells isroughly ten-fold lower. Alterations in antibodies that increase the ADCCactivity of an antibody relative to its parent antibody are known in theart, and generally correlate with modifications that increase thebinding affinity of the variant antibody to FcγRIII (see for example,U.S. Pat. No. 6,737,056). For example, mutations are introduced into theFc region at one or more positions (with reference to their position inIgGy1) selected from 256, 290, 298, 312, 326, 330, 333, 334, 339, 360,378 and 430 (numbering according to Kabat et al. Sequences of Proteinsof Immunological Interest, 1991). Preferred mutations are at one or morepositions selected from 298, 333, and 334. For example, alaninesubstitutions may be introduced.

ADCC activity of the antibody is also influenced by the particular cellline used to produce the antibody. For example, antibodies produced inthe mouse myeloma NS/0 cells (or SP2/0 cells) generally have low ADCC,and antibodies produced in rat myeloma YO cells (or YB2/0) cells havehigh ADCC (Lifely et al., (1995) Glycobiology 5:813-822). It is known inthe art that the type of cell line used for antibody expression affectsthe carbohydrate structure of the N-linked glycosyl chain, which isattached to the Fc region of the antibody at position corresponding toN297 in IgGγ1. The carbohydrate structure of antibodies produced in CHOcells is fucosylated, whereas the carbohydrate chain of antibodiesproduced in YB2/0 is largely absent of fucose (Shinkawa et al., (2003)JBC 278:3466-3473). Antibodies that lack fucose on the carbohydratestructure bind to human FcγRIIIa with higher affinity (Shields et al.,(2002) JBC 277:26733-26740). In certain embodiments, anti-CD19antibodies with variable regions of the invention are characterized byhaving reduced fucosylation on the N-linked glycosyl chain of the Fcportion of the antibody.

It is also often useful to alter the serum half-life of the antibody.The serum half-life of an antibody, as of an immunoglobulin fusionprotein, is influenced by the ability of that antibody to bind to an Fcreceptor (FcR) (Gillies et al., Cancer Research (1999) 59:2159-66). TheCH2 and CH3 domains of IgG2 and IgG4 have undetectable or reducedbinding affinity to Fc receptors compared to those of IgG1. Accordingly,the serum half-life of the featured antibody can be increased by usingthe CH2 and/or CH3 domain from IgG2 or IgG4 isotypes. Alternatively, theantibody can include a CH2 and/or CH3 domain from IgG1 or IgG3 withmodification in one or more amino acids in these domains to reduce thebinding affinity for Fc receptors (see, e.g., U.S. patent applicationSer. No. 09/256,156, published as U.S. patent application publication2003-0105294).

The hinge region of the Fc portion normally adjoins the C-terminus ofthe CH1 domain of the heavy chain constant region. When included in theproteins of the present invention, the hinge is homologous to anaturally-occurring immunoglobulin region and typically includescysteine residues linking two heavy chains via disulfide bonds as innatural immunoglobulins. Representative sequences of hinge regions forhuman and mouse immunoglobulin can be found in ANTIBODY ENGINEERING, aPRACTICAL GUIDE, (Borrebaeck, ed., W. H. Freeman and Co., 1992).

Suitable hinge regions for the present invention can be derived fromIgG1, IgG2, IgG3, IgG4, and other immunoglobulin isotypes. The IgG1isotype has two disulfide bonds in the hinge region permitting efficientand consistent disulfide bonding formation. Therefore, a preferred hingeregion of the present invention is derived from IgG1. Optionally, thefirst, most N-terminal cysteine of an IgG1 hinge is mutated to enhancethe expression and assembly of antibodies or antibody fusion proteins ofthe invention (see, e.g., U.S. patent application Ser. No. 10/093,958,published as U.S. patent application publication 2003-0044423).

In contrast to IgG1, the hinge region of IgG4 is known to forminterchain disulfide bonds inefficiently (Angal et al., (1993), Mol.Immunol. 30:105-8). Also, the IgG2 hinge region has four disulfide bondsthat tend to promote oligomerization and possibly incorrect disulfidebonding during secretion in recombinant systems. One suitable hingeregion for the present invention can be derived from the IgG4 hingeregion, preferentially containing a mutation that enhances correctformation of disulfide bonds between heavy chain-derived moieties (Angalet al., (1993), Mol. Immunol. 30(1):105-8). Another preferred hingeregion is derived from an IgG2 hinge in which the first two cysteinesare each mutated to another amino acid, such as, in order of generalpreference, serine, alanine, threonine, proline, glutamic acid,glutamine, lysine, histidine, arginine, asparagine, aspartic acid,glycine, methionine, valine, isoleucine, leucine, tyrosine,phenylalanine, tryptophan or selenocysteine (see, e.g., U.S. patentapplication publication 2003-0044423).

An Fc portion fused to an antibody variable region of the invention cancontain CH2 and/or CH3 domains and a hinge region that are derived fromdifferent antibody isotypes. For example, the Fc portion can contain CH2and/or CH3 domains of IgG2 or IgG4 and a hinge region of IgG1. Assemblyof such hybrid Fc portions has been described in U.S. patent applicationpublication 2003-0044423.

When fused to an antibody variable region of the invention, the Fcportion may contain one or more amino acid modifications that generallyextend the serum half-life of an Fc fusion protein. Such amino acidmodifications include mutations substantially decreasing or eliminatingFc receptor binding or complement fixing activity. For example, one typeof such mutation removes the glycosylation site of the Fc portion of animmunoglobulin heavy chain. In IgG1, the glycosylation site is Asn297(see, for example, U.S. patent application Ser. No. 10/310,719,published as U.S. patent application publication 2003-0166163).

The antibody variable regions of the invention can be attached to adiagnostic and/or a therapeutic agent. The agent can be fused to theantibody to produce a fusion protein. Alternatively, the agent can bechemically coupled to the antibody to produce an immuno-conjugate. Theagent can be, for example, a toxin, radiolabel, imaging agent,immunostimulatory moiety or the like.

The antibody variable region of the invention can be attached to acytokine. Preferred cytokines include interleukins such as interleukin-2(IL-2), IL-4, IL-5, IL-6, IL-7, IL-10, IL-12, IL-13, IL-14, IL-15,IL-16, IL-18, IL-21, and IL-23, hematopoietic factors such asgranulocyte-macrophage colony stimulating factor (GM-CSF), granulocytecolony stimulating factor (G-CSF) and erythropoietin, tumor necrosisfactors (TNF) such as TNFα, lymphokines such as lymphotoxin, regulatorsof metabolic processes such as leptin, interferons such as interferon α,interferon β, and interferon γ, and chemokines. Preferably, theantibody-cytokine fusion protein or immunoconjugate displays cytokinebiological activity. In one embodiment, the antibody variable domain isfused to IL-2. Preferably, several amino acids within the IL-2 moietyare mutated to reduce toxicity, as described in U.S. patent applicationpublication 2003-0166163.

Optionally, the protein complexes can further include a second agent,such as a second cytokine. In one embodiment, a B4 VHvx/VKvy antibodyfusion protein includes IL-12 and IL-2. The construction of proteincomplexes containing an immunoglobulin domain and two, differentcytokines is described in detail in U.S. Pat. No. 6,617,135.

Antibody Production

Antibodies of the invention, as well as other variable region-containingproteins of the invention, are produced by methods well known in the artof protein engineering. Nucleic acid vectors capable of expressing aheavy chain and a light chain which include sequences of the inventionare introduced into the appropriate cell and the recombinant proteinproduct is expressed and purified. For example, antibodies of theinvention can be produced in engineered mammalian cell lines such asNS/0 cells, CHO cells, SP2/0 cells (SP2/0-Ag14; ATCC-CRL 1581), YB2/0cells (YB2/3HL.P2.G11.16Ag.20; ATCC CRL-1662), or other mammalian cellswell known in the art of antibody production. In one embodiment, B4VHvx/VKvy antibodies are produced in NS/0 cells. In another embodiment,B4 VHvx/VKvy antibodies are produced in YB2/0 cells. Alternatively,yeast, plants, insect cells, or bacteria may be used to produce proteinscontaining variable regions of the invention.

Administration

The antibodies of the invention are preferably used to treat patientswith B cell disorders such as B cell lymphomas or autoimmune disorderswith a B cell component such as rheumatoid arthritis, myasthenia gravis,multiple sclerosis, systemic lupus erythematosus, and so on. For B celllymphoma, depending on the judgment of the physician, it may be usefulto treat a patient that has failed other therapies. For example, somepatients who are treated with Rituxan™ may initially respond, butRituxan™-resistant cancer cells may arise. Such patients shouldgenerally still respond to the antibodies of the invention.

In the case of antibodies directed against CD19, it is sometimes usefulto clear the normal B cells from the body, as these cells are likely totitrate the antibody of the invention. Rituxan™ may be used for thispurpose, according to standard procedures. Alternatively, the anti-CD 19antibodies of the invention may be used to clear the normal B cells fromthe body, for example as described in Example 5 and Example 9.

Infusion is the preferred method of administration. Other methods ofadministration include injection routes such as subcutaneous,intradermal, intramuscular, intraperitoneal, or intravenous (bolus)delivery. Inhalation and oral delivery are also possible methods ofdelivery.

For a 70 kilogram human, a typical dose is in the range of about 50milligrams to 2 grams, with a preferred dose in the range of about400-600 milligrams. Dosing may be repeated about once every three to sixweeks, for example, during which normal B cells and tumor cells aremonitored.

Fusion proteins of the present invention are useful in treating humandisease, such as cancer. When treating cancer, it is for example usefulto administer an antibody-IL-2 fusion protein comprising the variableregions of the invention by infusion or subcutaneous injection, usingdoses of 0.1 to 100 milligrams/meter²/patient. In a preferredembodiment, it is particularly useful to administer an antibody-IL-2fusion protein comprising the variable regions of the invention byinfusion or subcutaneous injection, using doses of 1 to 10milligrams/meter²/patient, and more preferably about 3 to 6milligrams/meter²/patient.

Pharmaceutical compositions of the invention may be used in the form ofsolid, semisolid, or liquid dosage forms, such as, for example, pills,capsules, powders, liquids, suspensions, or the like, preferably in unitdosage forms suitable for administration of precise dosages. Thecompositions include a conventional pharmaceutical carrier or excipientand, in addition, may include other medicinal agents, pharmaceuticalagents, carriers, adjuvants, etc. Such excipients may include otherproteins, such as, for example, human serum albumin or plasma proteins.Actual methods of preparing such dosage forms are known or will beapparent to those skilled in the art. The composition or formulation tobe administered will, in any event, contain a quantity of the activecomponent(s) in an amount effective to achieve the desired effect in thesubject being treated.

Administration of the compositions hereof can be via any of the acceptedmodes of administration for agents that exhibit such activity. Thesemethods local or systemic administration. Intravenous injection in apharmaceutically acceptable carrier is a preferred method ofadministration. The amount of active compound administered will, ofcourse, be dependent on the subject being treated, the severity of theaffliction, the manner of administration, and the judgment of theprescribing physician.

The invention is further illustrated through the following non-limitingexamples.

Example 1 Construction of Anti-CD19 Antibodies Containing VariableRegion Heavy and Light Chains of the Invention

Standard genetic engineering techniques were used to introduce nucleicacid sequences encoding a heavy chain region and light chain region ofthe invention into an appropriate mammalian expression vector. Exemplarycloning strategies are described below. The expression vector pdHL12 isa later-generation pdHL expression vector engineered to contain uniquerestriction sites for the insertion of nucleic acid cassettes encodingheavy and light chain variable regions. pdHL12 is designed to acceptnucleic acids encoding the heavy chain variable region as a Nhe I/HindIII fragment, and nucleic acids encoding the light chain variable regionas an Afl II/Bam HI fragment, and to co-express intact antibody heavyand light chains (see, for example US patent application 2003/0157054).

Nucleic acid sequences of heavy chain variable regions of the invention,flanked by sequences with endonuclease restriction recognition sequences5′-CTTAAGC-3′ (upstream, containing the Nhe I site) and5′-CGTAAGTGGATCC-3′ (downstream, containing the Hind III site), weresynthesized de novo and inserted into a pUC vector-derived carrierplasmid (Blue Heron Biotechnology, Bothell, Wash.). The nucleic acid wasexcised from the carrier plasmid as a Nhe I/Hind DI fragment and ligatedto the appropriate vector fragment of a likewise digested pdHL12plasmid. Nucleic acid sequences for B4 VH0 (SEQ ID NO:1), B4 VHv1 (SEQID NO:2), B4 VHv2 (SEQ ID NO:3), B4 VHv3 (SEQ ID NO:4), B4 VHv4 (SEQ IDNO:5), B4 VHv5 (SEQ ID NO:6), and B4 VHv6 (SEQ ID NO:7) are shown.

Similarly, nucleic acids of light chain variable regions of theinvention, flanked by sequences with endonuclease restrictionrecognition sequences 5′-GCTAGCTCCAGC-3′ (upstream, containing the AflII site) and 5′-GGTAAGCTT-3′ (downstream, containing the Bam HI site),were synthesized de novo and inserted into a pUC vector-derived carrierplasmid (Blue Heron Biotechnology, Bothell, Wash.). The nucleic acid wasexcised from the carrier plasmid as an Afl H/Bam HI fragment and ligatedto the appropriate vector fragment of a likewise digested pdHL12plasmid. Nucleic acid sequences encoding B4 VK0 (SEQ ID NO:8), B4 VKv1(SEQ ID NO:9), B4 VKv2 (SEQ ID NO:10), B4 VKv3 (SEQ ID NO:11), and B4VKv4 (SEQ ID NO:12) are shown.

By inserting the nucleic acid sequences encoding the different heavy andlight chain variable regions combinatorially into pdHL12, a panel ofplasmids encoding B4 antibodies of the invention, B4 VHvx/VKvy, weregenerated.

Example 2 Expression and Purification of Antibodies of the Invention

The following general techniques are used in the subsequent Examples.

1A. Cell Culture and Transfection

To express antibodies transiently from mammalian cells, plasmid DNA isintroduced into human embryonic kidney 293 cells, or baby hamster kidney(BHK) cells, by co-precipitation of plasmid DNA with calcium phosphateand cells are grown without selection for plasmid maintenance [Sambrooket al. (1989) Molecular Cloning: A Laboratory Manual, Cold SpringHarbor, N.Y.].

Stably transfected clones are obtained by one of several standardmethods, for example, by electroporation or by nucleofection.Electroporation of DNA into mouse myeloma NS/0 cells is performed asfollows. NS/0 cells are grown in Dulbecco's modified Eagle's medium(DMEM, Life Technologies) supplemented with 10% fetal bovine serum, 2 mMglutamine, 1 mM sodium pyruvate and 1× penicillin/streptomycin. About5×10⁶ cells are washed once with PBS and resuspended in 0.5 ml phosphatebuffer solution (PBS). Ten micrograms of linearized plasmid DNA is thenincubated with the cells in a Gene Pulser Cuvette (0.4 cm electrode gap,BioRad) for 10 minutes on ice. Electroporation is performed using a GenePulser (BioRad) with settings at 0.25 V and microF. Cells are allowed torecover for 10 minutes on ice, after which they are resuspended ingrowth medium and then plated onto two 96-well plates. Stablytransfected clones are selected by growth in the presence of 100 nMmethotrexate (MTX), which is introduced two days post-transfection. Thecells are fed every 3 days for two to three more times, andMTX-resistant clones generally appeared in 2 to 3 weeks. Supernatantsfrom clones are assayed by anti-human Fc ELISA to identify highproducers [Gillies et al. (1989) J. Immunol. Methods 125:191]. Highproducing clones are isolated and propagated in growth medium containing100 nM MTX.

Similarly, other cell lines may be used to obtain stably transfectedclones by essentially the same method, such as CHO cells, BHK cells,SP2/0 cells, and YB2/0 cells. When YB2/0 cells were used, stablytransfected clones were generally selected by growth in the presence of50 nM MTX.

Stably transfected clones, for example from rat myeloma YB2/0 cells,were also obtained by nucleofection. About 2×10⁶YB2/0 cells, grown inDulbecco's modified Eagle's medium (DMEM) supplemented with heatinactivated 10% fetal bovine serum, 2 mM glutamine, 1 mM sodiumpyruvate, and 1× penicillin/streptomycin, were centrifuged at 90×g atroom temperature for 10 min and resuspended in 100 μl of supplementedNucleofector Solution V. 100 μl of the cell suspension was mixed with 2μg of linearized plasmid DNA (linearized at the Fsp I site in theβ-lactamase sequence), transferred into a cuvette (Amaxa), and thenucleofection was performed using the appropriate Nucleofector (Amaxa)program, Q-20. 500 μl pre-warmed culture medium was added and the samplewas transferred into a well of a 12-well plate. One day posttransfection, the cells were resuspended in growth medium and platedonto 96 well plates at cell densities ranging from approximately 10cells/well to approximately 600 cells/well. Stably transfected cloneswere selected by growth in the presence of 50 nM methotrexate (MTX),which was introduced two days post-transfection. The cells were fedevery 2 or 3 days twice more, and MTX-resistant clones generallyappeared in 2 to 3 weeks. Supernatants from clones were assayed byanti-human Fc ELISA to identify high producers [Gillies et al. (1989) J.Immunol. Methods 125:191]. High producing clones were isolated andpropagated in growth medium containing 50 nM MTX.

The cells may be grown in an alternate medium known in the art, such asHSFM supplemented with 2.5% fetal bovine serum and 100 nM methotrexate,or a protein free medium such as CD medium.

1B. ELISAs

Different ELISAs are used to determine the concentrations of proteinproducts in the supernatants of MTX-resistant clones and other testsamples. For example, the anti-huFc ELISA is used to measure the amountof human Fc-containing proteins, e.g., chimeric antibodies, and theanti-hu kappa ELISA is used to measure the amount of kappa light chain(of chimeric or human immunoglobulins).

The anti-huFc ELISA is described in detail below.

A. Coating Plates

ELISA plates are coated with AffiniPure goat anti-human IgG (H+ L)(Jackson Immuno Research) at 5 microgram/ml in PBS, and 100 μl/well in96-well plates (Nunc-Immuno plate Maxisorp). Coated plates are coveredand incubated at 4° C. overnight. Plates are then washed 4 times with0.05% Tween (Tween 20) in PBS, and blocked with 1% BSA/1% goat serum inPBS, 200 microliter/well. After incubation with the blocking buffer at37° C. for 2 hours, the plates are washed 4 times with 0.05% Tween inPBS and tapped dry on paper towels.

B. Incubation with Test Samples and Secondary Antibody

Test samples are diluted to the proper concentrations in sample buffer,which contains 1% BSA/1% goat serum/0.05% Tween in PBS. A standard curveis prepared with a chimeric antibody (with a human Fc), theconcentration of which is known. To prepare a standard curve, serialdilutions are made in the sample buffer to give a standard curve rangingfrom 125 ng/ml to 3.9 ng/ml. The diluted samples and standards are addedto the plate, 100 microliter/well, and the plate is incubated at 37° C.for 2 hours.

After incubation, the plate is washed 8 times with 0.05% Tween in PBS.To each well is then added 100 microliter of the secondary antibody, thehorseradish peroxidase (HRP)-conjugated anti-human IgG (Jackson ImmunoResearch), diluted around 1:120,000 in the sample buffer. The exactdilution of the secondary antibody has to be determined for each lot ofthe HRP-conjugated anti-human IgG. After incubation at 37° C. for 2hours, the plate is washed 8 times with 0.05% Tween in PBS.

C. Development

The substrate solution is added to the plate at 100 μl/well. Thesubstrate solution is prepared by dissolving 30 mg of o-phenylenediaminedihydrochloride (OPD) (1 tablet) into 15 ml of 0.025 M citric acid/0.05MNa₂HPO₄ buffer, pH 5, which contains 0.03% of freshly added H₂O₂. Thecolor is allowed to develop for 30 minutes at room temperature in thedark. The developing time is subject to change, depending on lot to lotvariability of the coated plates, the secondary antibody, etc. The colordevelopment in the standard curve is observed to determine when to stopthe reaction. The reaction is stopped by adding 4N H₂SO₄, 100 μl/well.The plate is read by a plate reader, which is set at both 490 nm and 650nm and programmed to subtract off the background OD at 650 nm from theOD at 490 nm.

The anti-hu kappa ELISA follows the same procedure as described above,except that the secondary antibody used is horseradishperoxidase-conjugated goat anti-hu kappa (Southern Biotechnology Assoc.Inc., Birmingham, Ala.), used at a 1:4000 dilution.

Purification

Standard antibody purification procedures were followed. Typically, B4VHvx/VKvy antibody compositions of the invention were purified fromcell-culture supernatants via Protein A chromatography based on theaffinity of the Fc portion for Protein A. The conditioned supernatantfrom cells expressing B4 VHvx/VKvy antibody compositions was loaded ontoa pre-equilibrated Fast-Flow Protein A Sepharose column. The column waswashed extensively with sodium phosphate buffer (50 mM Sodium Phosphate,150 mM NaCl at neutral pH). Bound protein was eluted by a low pH (pH2.5-3) sodium phosphate buffer (composition as above) and the elutedfractions were immediately neutralized to about pH 6.5 with 1M Trisbase. The compositions were stored in 50 mM Sodium Phosphate, 150 mMNaCl, pH 6.5 supplemented with Tween 80 to 0.01%.

The purity and integrity of the product was routinely assessed by HPLCsize exclusion chromatography and by SDS-PAGE. Results showed that theB4 VHvx/VKvy antibodies of the invention were typically greater than 90%non-aggregated and intact, with little evidence of degradation products.

Example 3 Determination of the Relative Binding Affinity of B4Antibodies of the Invention to CD-19 Presenting Cells

To ascertain that the antibodies of the invention retained binding toCD19 a competition assay was used, in which the strength of theseantibodies to inhibit binding of labeled parental B4 antibody (B4VH0/VK0) to Daudi lymphoma cells, which bear the CD19 antigen, wasmeasured.

Biotin-labeled B4 VH0/VK0 antibody was prepared using the EZ-linkSulfo-NHS-LC-Biotinylation Kit (Pierce, #21430) according to thesupplied protocol. The product was dialyzed with a Slide-a-lyzer(Pierce, #66425), and analyzed by HPLC size exclusion chromatography.

Briefly, a titration series was prepared of biotin-labeled B4 VH0/VK0antibody pre-mixed at a final concentration of 100 ng/ml with one of theunlabeled, experimental B4 VHvx/VKvy antibodies at 800 ng/ml, 400 ng/ml,200 ng/ml, 100 ng/ml, and 50 ng/ml in a PBS/2% serum buffer. As acontrol, the biotin-labeled antibody was pre-mixed with unlabeled B4VH0/VK0 antibody at the same concentrations as above (inhibitioncontrol) or with buffer only (positive binding control). The combinedantibodies were added to Daudi cells for 30 minutes at 4° C. A 1:200dilution of FITC-labeled streptavidin was added to the cells and thesamples were incubated for a further 30 minutes at 4° C. Bound amount oflabeled B4 VH0/VK0 antibody was quantitated by FACS analysis, and theresults were expressed as “percent inhibition,” relative to the positivebinding control. The tested antibodies were B4 VH0/VK0 (C), B4 VHv1/VKv1(1), B4 VHv2/VKv1 (2), B4 VHv1/VKv2 (3), B4 VHv2/VKv2 (4), B4 VHv3/VKv3(5), B4 VHv4/VKv3 (6), B4 VHv3/VKv4 (7), B4 VHv4/VKv4 (8), B4 VHv5/VKv4(9), and B4 VHv6/VKv4 (10). Representative results of two experimentsare shown in Table 3 and Table 4.

TABLE 3 Inhibition of Biotin-B4 VH0/VK0 binding to Daudi cells byantibodies of the invention. Antibody Ratio (% inhibition of labeled B4binding) (unlabeledl/labeled) C 1 2 3 4 5 6 7 8 8x 68 56 56 56 56 60 5660 60 4x 56 44 40 44 44 40 44 52 48 2x 44 28 28 32 32 36 32 36 32 1x 2816 16 20 16 20 16 20 16 0.5x   16 12 8 12 8 12 12 12 12

TABLE 4 Inhibition of Biotin-B4 VH0/VK0 binding to Daudi cells byantibodies of the invention. Ratio Antibody (unlabeledl/ (% inhibitionof labeled B4 binding) labeled) C 9 10 8x 93 90 87 4x 90 87 80 2x 83 7070 1x 67 57 60 0.5x 50 47 53

As shown in Table 3 and Table 4, it was found that the B4 VHvx/VKvyantibodies inhibited binding of labeled B4 antibody to Daudi cells to asimilar extent as the unlabeled B4 VH0/VK0 antibody did, indicating thatthe affinities of the B4 VHvx/VKvy antibodies and B4 VH0/VK0 antibodyare similar.

Example 4 ADCC Activity of Antibodies of the Invention

ADCC activity mediated by the antibodies of the invention produced invarious cell lines was assessed. ADCC was determined by a standard ⁵¹Crrelease assay, as practiced in the art. A serial dilution of theantibodies was prepared (4-fold dilutions in a range from 100 ng/ml to0.025 ng/ml), and lysis by purified human PBMCs (effector cells) of⁵¹Cr-labeled Daudi cells (target; E:T is 100:1) in the presence of theantibodies was measured by specific ⁵¹Cr release, relative to totalcellular ⁵¹Cr (adjusting for spontaneously released ⁵¹Cr). B4 VHv4NKv4antibodies produced from human embryonic kidney 293T cells or from YB2/0cells, and B4 VHv5NKv4 antibodies produced from a NS/0 cell line or fromYB2/0 cells were tested.

FIG. 38 shows the result of such an experiment. Both antibodies obtainedfrom YB2/0 cell expression were equally active in mediating ADCC, and atleast 50 fold more active than the corresponding antibody obtained byexpression from a NS/0 cell line or from 293T cells. B4 VHv4NKv4produced from 293T cells was more active than B4 VHv5/VKv4 produced froma NS/0 cell line.

Example 5 Depletion of Human B Cells Grafted into SCID Mice byAntibodies of the Invention

The depletion of B cells is useful in a number of therapeutic contexts.For example, antibody-driven autoimmune and inflammatory disorders maybe treated with antibodies of the invention to reduce or essentiallyeliminate B cells. Alternatively, when treating with a tumor-targetingagent such as Zevalin™ or Bexxar™ or a Leu16-IL2 fusion protein(WO2005/016969), it is useful to first eliminate normal B cells.

To address whether an antibody of the invention could be used to depletehuman B cells, the following experiment was performed. On day 0, maleSCID CB17 mice (n=3) were engrafted with purified human PBMCs in whichabout 4.5×10⁷ cells in 0.2 mls of PBS were injected intraperitoneally,essentially following a protocol described for the transfer of humanspleen cells (Yacoub-Youssef et al., Transpl. Immunol. (2005)15:157-164). On day 3, the mice were injected intraperitoneally witheither PBS or 50 micrograms of the anti-CD20 antibody Leu16 or with theK4H4 anti-CD19 antibody. Levels of human IgM were measured by a humanIgM ELISA quantitation kit (Bethyl Laboratories; Cat # E80-100) on days7, 14, and 21.

FIG. 39 shows typical results. In the PBS-treated controls, the titer ofhuman IgM steadily increased, reaching about 800 micrograms/ml on day42. In the mice treated with either Leu16 or B4 VHv5/VKv4 antibody,human IgM titers were essentially absent, indicating that human B cellswere depleted by these antibody treatments.

Example 6 Treatment of a Lymphoma-Bearing Mammal with an Antibody of theInvention

To address whether the antibodies of the invention were functional invivo, the B4 VHv4/VKv4 antibody, expressed in YB2/0 cells, was tested inan animal model of human lymphoma. Eight-week-old SCID CB 17 mice (n=6)were injected intravenously with about 1×10⁶ viable ‘Namalwa’Nalm-6-UM-1 cells on day 0. On days 1, 3 and 5, mice were treated with500 micrograms of antibody intraperitoneally or with PBS. About one totwo times per week the mice were examined to see which mice had becomeill enough to require euthanasia.

FIG. 40 shows typical results of such an experiment. Mice treated withPBS all became ill within 10 weeks of the injection of the tumor cells,while mice treated with the B4 VHv4/VKv4 antibody became ill at latertimes, and three of the six mice in this group remained healthythroughout the 30-week course of the experiment.

Example 7 Treatment of a Burkitt's Lymphoma-Bearing Mammal with anAntibody of the Invention in Combination with Chemotherapy

To address whether the antibodies of the invention could be used in vivoin combination with chemotherapy, the B4 VHv4NKv4 antibody was tested inanimal models of human lymphoma that were more stringent than in theprevious example, corresponding to more advanced or harder to treatforms of lymphoma. In one representative experiment, the Daudi cellline, which is a Burkitt's lymphoma cell line, was treated with the B4VHv4NKv4 antibody, expressed in YB2/0 cells, with or withoutcyclophosphamide (CPA). Eight-week-old SCID CB 17 mice (n=6) wereinjected intravenously with about 5×10⁶ viable Daudi cells on day 0. Ondays 8 and 12, mice were treated with 100 micrograms of antibody or PBS.On day 7, the mice were treated with PBS or 75 micrograms of CPA perkilogram of body weight. Treatments were administered intraperitoneallyin 0.2 mls. About one to two times per week the mice were examined tosee which mice had become ill enough to require euthanasia.

FIG. 41 shows the results of a typical experiment. In the control grouptreated with neither antibody nor CPA, all of the mice became ill withinfour weeks of injection of the lymphoma cells. In the groups of micesingly treated with either the B4 VHv4/VKv4 antibody or CPA, 5 out ofthe 6 mice were healthy for at least five weeks but became ill withinabout six weeks. In the group of mice treated with both B4 VHv4/VKv4antibody and CPA, all of the mice remained healthy for at least eightweeks.

Example 8 Treatment of Lymphoma Disseminated Disease with an Antibody ofthe Invention Combined with Chemotherapy

In another set of experiments, Namalwa cells were injected intravenouslyinto mice as described in the previous Example, except that 2×10⁶ cellswere used instead of 1×10⁶ cells. This increased number of cells resultsin a more aggressive disease course, as indicated by a comparison of thePBS-treated mice in FIG. 42, which all became ill within three weeks, asopposed to the PBS-treated mice in FIG. 40, which became ill betweenfive and 10 weeks. Mice (n=5) were treated with 500 micrograms ofantibody intraperitoneally or with PBS on days 3, 7 and 11. Mice werealso treated with either cyclophosphamide (75 mg/kg, i.p.), orvincristine (0.4 mg/kg, i.v.) or doxorubicin (3 mg/kg, i.v.) or PBS(i.v.) on days 3, 7, and 11. The results are shown in FIG. 42( a-c). Theresults indicated that each of the three chemotherapeutic agents couldbe combined with an antibody of the invention.

Example 9 Treatment of a Human Patient with Antibodies and Methods ofthe Invention

The anti-CD19 antibodies of the invention are used to treat humandiseases and disorders as follows. In general, the preferred method ofadministration is by intravenous infusion or intravenous injection,although subcutaneous injection, inhalation, oral delivery, and othermethods are also possible. Administration about once every 2, 3 or 4weeks is used, although the frequency of administration may varydepending on the needs of the patient. A typical dose is about 100 to800 mgs for an adult human. Treated patients are monitored for signs ofinfection that may result from immunosuppression.

For example, a patient with Castleman's disease is treated with theanti-CD19 B4 VHv4/VKv4 antibody of the invention about once every twoweeks at a dose of about 8 mg/kg, with administration by drip infusionfor about 1 hour.

A patient with rheumatoid arthritis is treated with the anti-CD19 B4VHv4/VKv4 antibody about once every four weeks at a dose of about 8mg/kg, with administration by drip infusion for about 1 hour.Progression of joint destruction is found to be significantly inhibitedby monotherapy, even when compared to disease-modifying anti-rheumaticdrugs.

A patient with Crohn's disease is treated with the anti-CD19 B4VHv4/VKv4 antibody about once every four weeks at a dose of about 8mg/kg, with administration by drip infusion for about 1 hour.

A patient with multiple myeloma is treated with the anti-CD19 B4VHv4/VKv4 antibody about once every three weeks at a dose of about 8mg/kg, with administration by drip infusion for about 1 hour. Treatmentwith the anti-CD19 B4 VHv4NKv4 is combined with a standard-of-caretreatment for multiple myeloma as determined by a physician asappropriate for the patient.

A patient with a B cell lymphoma is treated with the anti-CD19 B4VHv4NKv4 antibody about once every three weeks at a dose of about 8mg/kg, with administration by drip infusion for about 1 hour, optionallyin combination with an antibody such as Rituxan™ at about 375 milligramsper square meter of body surface area, which is administered every week,or with the anti-CD22 antibody epratuzumab. Alternatively, in the caseof a patient with refractory lymphoma, treatment with the anti-CD19 B4VHv4NKv4 antibody is combined with a radioimmunoconjugate such asBexxar™ or Zevalin™.

More specifically, a patient with a B cell lymphoma is treated with theanti-CD19 B4 VHv4/VKv4 antibody about once every three weeks at a doseof about 8 mg/kg, with administration by drip infusion for about 1 hour,optionally in combination with a chemotherapeutic regimen such ascyclophosphamide plus vincristine plus doxorubicin plus prednisolone(“CHOP”), or CHOP plus bleomycin, or CHOP plus etoposide, ormitoxantrone plus vincristine plus thiotepa, or etoposide plusprednisolone plus cytarabin plus cisplatin, or mesna plus ifosfamideplus mitoxantrone plus etoposide, or bendamustin, or fludaribin and2-CdA.

In an alternative treatment strategy, a patient with a B cell lymphomais initially treated with the anti-CD19 B4 VHv4/VKv4 antibody at a doseof about 8 mg/kg, and is then later treated with an anti-CD20-IL2 fusionprotein such as that described in WO2005/016969. For example, a patientis treated on day 1 with the anti-CD19 B4 VHv4NKv4 antibody withadministration by drip infusion for about 1 hour, and then treated onday 2 and day 4 with an anti-CD20-IL2 fusion protein at a dose of about150 micrograms per kg with administration by drip infusion for about 4hours, and this cycle is repeated about every 3 weeks. Without wishingto be bound by theory, the anti-CD19 B4 VHv4/VKv4 antibody has theeffect of clearing most of the normal B cells from the patient, so thatthe anti-CD20-IL2 fusion protein exerts its effects by binding toremaining tumor cells.

EQUIVALENTS

The invention may be embodied in other specific forms without departingfrom the spirit or essential characteristics thereof. The foregoingembodiments are therefore to be considered in all respects illustrativerather than limiting on the invention described herein. Scope of theinvention is thus indicated by the appended claims rather than by theforegoing description, and all changes which come within the meaning andrange of equivalency of the claims are intended to be embraced therein.

1-22. (canceled)
 23. A nucleic acid encoding an anti-CD 19 antibodyvariable domain comprising a heavy chain variable region that is atleast 90% identical to SEQ ID NO: 13 and has an amino acid substitutionat one or more residues corresponding to Gln5, Arg19, Leu20, Arg40,Gln43, Lys65, Ser85, Ser88, and Val93.
 24. The nucleic acid of claim 23,the heavy chain variable region having, compared to SEQ ID NO:13, one ormore amino acid substitutions selected from the group consisting ofGln5Glu, Arg19Lys, Leu20Val, Arg40Thr, Gln43Lys, Lys65Asp, Ser85Asp,Ser88Ala, and Val93Thr.
 25. The nucleic acid of claim 23, wherein theheavy chain variable region is SEQ ID NO: 13 comprising an amino acidsubstitution at one or more residues corresponding to Gln5, Arg19,Leu20, Arg40, Gln43, Lys65, Ser85, Ser88, and Val93.
 26. The nucleicacid of claim 25, wherein the heavy chain variable region has one ormore amino acid substitutions selected from the group consisting ofGln5Glu, Arg19Lys, Leu20Val, Arg40Thr, Gln43Lys, Lys65Asp, Ser85Asp,Ser88Ala, and Val93Thr.
 27. The nucleic acid of claim 26, wherein theheavy chain variable region is the amino acid sequence of SEQ ID NO: 17.28. The nucleic acid of claim 23, further comprising a nucleic acidencoding a light chain variable region that is at least 90% identical toSEQ ID NO: 25 and has an amino acid substitution at one or more residuescorresponding to Ile10, Met11, Val19, Ser51, and Leu53.
 29. The nucleicacid of claim 28, wherein the light chain variable region is at least95% identical to SEQ ID NO:
 25. 30. A nucleic acid encoding an anti-CD19 antibody variable domain comprising a light chain variable regionthat is at least 90% identical to SEQ ID NO: 25 and has an amino acidsubstitution at one or more residues corresponding to Ile10, Met11,Val19, Ser51, and Leu53.
 31. The nucleic acid of claim 30, wherein thelight chain variable region has one or more amino acid substitutionsselected from the group consisting of Ile10Thr, Met11Leu, Val19Ala,Ser51Asp, and Leu53Thr.
 32. The nucleic acid of claim 30, wherein thelight chain variable region is SEQ ID NO: 25 comprising an amino acidsubstitution at one or more residues corresponding to Ile10, Met11,Val19, Ser51, and Leu53.
 33. The nucleic acid of claim 32, wherein thelight chain variable region has one or more amino acid substitutionsselected from the group consisting of Ile10Thr, Met11Leu, Val19Ala,Ser51Asp, and Leu53Thr.
 34. The nucleic acid of claim 33, wherein thelight chain variable region is the amino acid sequence of SEQ ID NO: 29.35. A nucleic acid encoding an anti-CD19 antibody variable domaincomprising: SEQ ID NO: 13 with an amino acid substitution at one or moreresidues corresponding to Gln5, Arg19, Leu20, Arg40, Gln43, Lys65,Ser85, Ser88, and Val93; and SEQ ID NO: 25 with an amino acidsubstitution at one or more residues corresponding to Ile10, Met11,Val19, Ser51, and Leu53.
 36. The nucleic acid of claim 35, wherein theheavy chain variable region comprises one or more of substitutionsGln5Glu, Arg19Lys, Leu20Val, Arg40Thr, Gln43Lys, Lys65Asp, Ser85Asp,Ser88Ala, and Val93Thr.
 37. The nucleic acid of claim 35, wherein thelight chain variable region comprises one or more of substitutionsIle10Thr, Met11Leu, Val19Ala, Ser51Asp, and Leu53Thr.
 38. A nucleic acidencoding an anti-CD19 antibody variable domain comprising a heavy chainvariable region of SEQ ID NO: 17 and a nucleic acid encoding a lightchain variable region of SEQ ID NO:
 29. 39. A nucleic acid encoding afusion protein comprising the anti-CD19 antibody variable domain ofclaim
 23. 40. A nucleic acid encoding a fusion protein comprising theanti-CD19 antibody variable domain of claim
 24. 41. A nucleic acidencoding a fusion protein comprising the anti-CD19 antibody variabledomain of claim
 25. 42. A nucleic acid encoding a fusion proteincomprising the anti-CD19 antibody variable domain of claim
 26. 43. Anucleic acid encoding a fusion protein comprising the anti-CD19 antibodyvariable domain of claim
 27. 44. A nucleic acid encoding a fusionprotein comprising the anti-CD19 antibody variable domain of claim 28.45. A nucleic acid encoding a fusion protein comprising the anti-CD19antibody variable domain of claim
 29. 46. A nucleic acid encoding afusion protein comprising the anti-CD19 antibody variable domain ofclaim
 30. 47. A nucleic acid encoding a fusion protein comprising theanti-CD19 antibody variable domain of claim
 31. 48. A nucleic acidencoding a fusion protein comprising the anti-CD19 antibody variabledomain of claim
 32. 49. A nucleic acid encoding a fusion proteincomprising the anti-CD19 antibody variable domain of claim
 33. 50. Anucleic acid encoding a fusion protein comprising the anti-CD19 antibodyvariable domain of claim
 34. 51. A nucleic acid encoding a fusionprotein comprising the anti-CD19 antibody variable domain of claim 35.52. A nucleic acid encoding a fusion protein comprising the anti-CD19antibody variable domain of claim
 36. 53. A nucleic acid encoding afusion protein comprising the anti-CD19 antibody variable domain ofclaim
 37. 54. A nucleic acid encoding a fusion protein comprising theanti-CD19 antibody variable domain of claim 38.